There is often a need to store viable cells for extended periods of time. It is most preferred that the cells be held in a state of suspended animation, a state wherein cell metabolism is entirely stopped. When needed, the cells are reanimated and used. Cells such as spermatozoa, blood and blood products are amongst the cell types for which long term storage is desired.
A generally accepted way of storing cells in a state of suspended animation is cryopreservation. Most simply, a sample containing viable cells is quickly cooled, for example by immersion in liquid nitrogen, to freeze the sample. When thawed under carefully controlled conditions acceptable levels of post-thaw cell viability are achieved.
The greatest practical disadvantage of cryopreservation of cells involves the difficulties in maintaining the frozen state. Refrigeration at cryogenic temperatures of 198 K is necessarily expensive (excepting in the Antarctic) and not a perfect solution as biological activity does not cease until about 143 K. The bulk of the required refrigeration systems renders cryopreserved cells not easily transportable.
An additional method for storing cells in a state of suspended animation involves removing a significant proportion of water from the cells (drying). The greatest advantage of drying cells is that dry cells can potentially be stored at close to room temperatures without requiring bulky and expensive storage devices. Typically, cells are air-dried: the cells in solution are spread out in a thin layer and water allowed to evaporate. Although clearly there is no danger of ice crystal formation damaging the cells, the fact that the drying process occurs at a temperature of at least above. 273 K where cell metabolism is active is a great disadvantage. Further, air drying has not been shown to be a practical method for the preservation of large-volume samples. Post-rehydration viability of air-dried cells is incidental at best. Apparently, either the air-drying process or the rehydration process causes cytolysis.
A number of researchers have found methods of improving post-rehydration viability of air-dried cells by the addition of cell membrane stabilizers. Such stabilizers added to a buffered cell-containing solution improve post-rehydration viability.
Another approach for drying cells involves freeze-drying. The simplest methods involve lyophilization of a frozen sample. Post-rehydration viability is poor.
It has been found that an effective methods of increasing post-thaw or post-rehydration viability of frozen or freeze-dried cells, respectively, is the addition of cryoprotectants, substances that protect cell membranes from the effects of freezing.
One group of cryoprotectants, termed permeating cryoprotectants are antifreeze molecules and includes dimethyl sulfoxide (DMSO), glycerol and related polyhydril alcohols (e.g. propylene or ethylene glycol).
Another group of cryoprotectants termed non-permeating or extracellular cryoprotectants, bind to membrane lipids. The membrane lipids are stabilized, thus preserving cell-structure integrity. Such cryoprotectants include proline and sugars (e.g. glucose). Crowe (in U.S. Pat. No. 4,857,319) has taught that trehalose is an exceptionally good cryoprotectant.
The disadvantages of using cryoprotectants are manifold. Different cell types require different cryoprotectants at different concentrations. Thus the use of cryoprotectants is not a general method but rather requires a complex and time-consuming optimization step. Second, rehydration and thawing must be very carefully due to the effect of osmotic pressure resulting from the presence of cryoprotectants in solution. Lastly, upon rehydration or thawing, the cells are found in an “unnatural” solution which must be purified or modified before being used.
Kusakabe et al. (“Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa” Proc. Natl. Acad. Sci., U.S.A. Nov. 20, 2001, 98(24), 13501-13506) has convincingly demonstrated that freeze-drying is a potentially successful strategy for the long term storage of cells. Mouse spermatozoa in a buffered solution without cryoprotectants were freeze dried by immersion in liquid nitrogen followed by 4 hours lyophilization. The freeze dried samples were stored at 277 K for up to 56 days. The results indicate a high level of DNA integrity after rehydration. However, the usefulness of the teachings of Kusakabe et al. is limited by the fact that the cells were lyophilized in 0.1 ml portions. One skilled in the art would remain unconvinced of the potential of this method for the preservation of cells on a larger scale.
In U.S. Pat. No. 5,873,254. which is incorporated by reference for all purposes as if fully set forth herein, is described an innovative method for the freezing of biological samples by directional cooling. Schematically depicted in FIG. 1 is a device 10 used to realize the teachings of U.S. Pat. No. 5,873.254. In FIG. 1A, a vessel 12 holding a biological sample 14 lays on a track 16. Track 16 leads from the surroundings into a cooling device 18. Cooling device 18 is substantially a block of a thermally conductive material having a tunnel 20 through which vessel 12 can pass when guided along track 16. In cooling device 18 is maintained a temperature gradient by the use of a plurality of cooling elements 22a, 22b and 22c. The temperature gradient is oriented so that there is a temperature gradient parallel to tunnel 20. The temperature of cooling device 18 at entrance 24 of tunnel 20 is typically roughly the temperature of sample 14 at the beginning of the freezing process. Exit 26 of tunnel 20 is cooler than entrance 24. A toothed rod 28, a gear 30, configured to engage rod 28, and an electrical motor 32, configured to rotate gear 30, are all elements of a mechanism configured to push vessel 12 into and through tunnel 20 at a controlled rate.
When it is desired to freeze biological sample 14, motor 32 is activated causing rod 28 to push vessel 12 into cooling device 18 through tunnel 20, FIG. 1B. Throughout the travel of biological sample 14 through tunnel 20, each portion of biological sample is cooled at a similar rate, but at any given moment the temperature of biological sample near the leading end 34 of vessel 12 is lower then that of biological sample near the following end 36 of vessel 12. A cooling front is formed inside biological sample 14, the cooling front traveling from leading end 34 towards following end 36, in parallel to the motion of vessel 12 along track 16. Biological sample 14 found in a given cross-section perpendicular to the motion of vessel 12 along track 16 is substantially homogenous in temperature. Through biological sample 14 is a temperature gradient parallel to track 16, the temperature gradient substantially mirroring the temperature gradient found along track 16.
Practically, it has been found that to prevent supercooling of biological sample 14 (and the concomitant uncontrolled and inhomogeneous freezing rate, see U.S. Pat. No. 5,873,254) it is necessary to “seed” biological sample near leading end 34 before entering tunnel 20. Seeding is done, for example, by applying liquid nitrogen to leading end 34.
An important parameter when applying the method of U.S. Pat. No. 5,873,254 is the cooling rate. The cooling rate C (in units of ° C./min) is determined by the temperature gradient G (in units of ° C./mm) alone track 16 and the rate of advancement of the cooling front P (in units of mm/sec) through biological sample 14, determined by the speed which vessel 12 travels through tunnel 20.
Cells in cryoprotectant-containing samples have exceptional post-thaw viability when frozen according to the method of U.S. Pat. No. 5,873,254, for example by using a device such as device 10. It is important to note that when a biological sample is lyophilized subsequent to freezing according to the method of U.S. Pat. No. 5,873,254, post-rehydration cell viability is insufficient.
A practical disadvantage of the method of U.S. Pat. No. 5,873,254 arises from the relatively limited amounts of sample that can be frozen at any one time. Experimentally it has been found that post-rehydration viability decreases when large volumes (generally greater than 1 ml) are frozen.
It would be highly advantageous to have a method of freezing large volume samples containing living cells. It would be exceptionally advantageous if such a method froze the sample in a way as to allow high post-rehydration post-lyophilization viability. It would be advantageous if such a method did not require the use of cryoprotectants.